Compounds for the treatment of cancer

ABSTRACT

It is shown that the potency of anti-cancer drugs, here exemplified by doxorubicin, can be increased by the use of polyunsaturated fatty acid amides and in particular specific combinations of such compounds, forming complexes with doxorubicin. Further, a modified form of doxorubicin is presented.

FIELD OF THE INVENTION

This invention relates to new compounds, which are useful in thetreatment of cancer. These compounds are used to increase the effect ofconventional cytotoxic pharmaceuticals.

BACKGROUND OF THE INVENTION

Cytostatic or cytotoxic compounds are widely used in the treatment ofcancer. Doxorubicin is an aminoglycosidic anthracycline antibiotic andwill be used as a typical representative of this group of compounds.

The cell membrane represents a physical barrier and there are somefactors that determine the rate of uptake of doxorubicin. The mainfactors are hydrophobicity (an increase will increase the rate ofuptake) and protonation degree of amino group—pKa (a decrease willincrease the rate of entry). The doxorubicin inhibits cell growth andhas a marked effect on the nuclear material, which becomesnon-specifically thickened, agglutinated or broken. The major bindingforce between doxorubicin and DNA is intercalation of the planarchromophore, stabilised by an external electrostatic binding of thepositive charged amino sugar residue with negative phosphate group ofDNA.

The intercalated drug molecules appear to prevent the changes inconformation of the helix, which are necessary as a preliminary toinitiation of nucleic acid synthesis. The major lethal effect ofdoxorubicin is inhibition of nucleic acid synthesis. As consequence thedrug is more active against dividing cells and the greatest effect is inthe S stage of the cell cycle (Brown J. R., Adriamycin and relatedanthracycline antibiotics in: Progress in Medicinal Chemistry edited byG. P. Ellis and G. B. West, Elsevier/North-Holland Biomedical Pressv.15, pp. 125-164, 1978).

Some observations are consistent with the formation complex ofelectrostatic nature between the positive amino group of doxorubicin andnegative phosphate group of phospholipids such as cardiolipin,phosphatidyl serine, phosphatidyl inositol and phosphatidic acid.Cardiolipin is an almost characteristic component of the inner membraneof mitochondria, which are abundant in the cardiac muscle. Thepathogenesis of the mitochondrial lesions is one of the major and morespecific sub-cellular changes characterizing doxorubicin cardiotoxicity.The rather selective toxicity doxorubicin for mitochondria may be due tothe high concentrations of cardiolipin in the mitochondria of thecardiac muscle (Duarte-Karim M., et al. Biochem. Biophys. Res. Comm.,v.71, N.2, pp. 658-663, 1976).

The interaction between doxorubicin and lipids has been studied usinglarge unilamellar vesicles (LUVET) composed of mixtures of anionicphospholipids and various zwitterionic phospholipids. Dilution ofanionic lipids with zwitterionic lipids leads to decreased membraneassociation of the drug because electrostatic forces are very importantin doxorubicin-membrane interaction. However, binding of doxorubicin toLUVET composed of anionic phospholipids combined withphosphatidylethanolamine (PE) is much higher than binding to LUVET madeof anionic lipids plus a range of other zwitterionic lipids such asphosphatidylcholine and the N-methyethanolamine andN,N-dimethylethanolamine derivatives of PE (Speelmans G, et al.,Biochemistry, v.36, N.28, pp. 8657-8662, 1997).

The interaction of adriamycin with human erythrocytes was investigatedin order to determine the membrane binding sites and the resultantstructural perturbation. Electron microscopy revealed that red bloodcells incubated with the therapeutic concentration of the drug in humanplasma changed their discoid shape to both stomatocytes and echinocytes.The drug was incubated with molecular models. One of them consisted ofdimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolaminemultilayers, representatives of phospholipid classes located in theouter and inner leaflets of the erythrocyte membrane, respectively.X-ray diffraction showed that adriamycin interaction perturbed the polarhead and acyl chain regions of both lipids. It is concluded thatadriamycin incorporates into both erythrocyte leaflets affecting itsmembrane structure (Suwalsky M., Z Naturforsch [C] v.54, N3-4, pp.271-277, 1999).

The different physicochemical properties ofdipalmitoylphosphatidylcholine liposomes with soybean-derived sterolshave been studied. Liposomal doxorubicin increased the pharmacologicaleffect compared with free drug, suggesting a decrease of side effect andlong circulation (Maitani Y., Yakugaku Zasshi, v.116, N.12, pp. 901-910,1996).

Liposomes containing polyethylene glycol-derivatised phospholipids areable to evade the reticulo-endothelial system and thereby remain incirculation for prolonged periods. The doxorubicin encapsulated in thesesterically stabilised liposomes suppresses the growth of establishedhuman lung tumour xenografts in severe combined immunodeficient mice andinhibits the spontaneous metastases of these tumours (Sakakibara T., etal., Cancer Res., v.56, N. 16, pp. 3743-3746, 1996).

A liposome encapsulation can protect surrounding tissue from thecytotoxic effects of the drugs after subcutaneous (s.c.) administration.Liposomes composed of “fluid-state” phospholipids only delayed thedamaging effects of doxorubicin when injected s.c. Liposomes with a morerigid nature were much more effective in preventing local tissue damageover a longer period of time when administered s.c. (Oussoren C., etal., Biochim. Biophys. Acta, v.1369, N.1, pp. 159-172, 1998).

Exogenous polyunsaturated fatty acids modulate the cytotoxic activity ofanti-cancer drugs in the human breast cancer cell line MDA-MB-231. Amongall polyunsaturated fatty acids tested, docosahexaenoic acid was themost potent in increasing doxorubicin cytotoxicity (E. Germain, et al.,Int. J. Cancer, v.75, pp. 578-583, 1998).

There remains a need for novel compounds and methods for the treatmentof cancer. The present invention aims i.a. to increase thepharmacological activity of presently used anti-cancer drugs, such asdoxorubicin, and to introduce novel approaches to the treatment ofcancer.

SHORT SUMMARY OF THE INVENTION

The present invention makes available new compounds and new combinationsof compounds, which, together with known cytotoxic or cytostaticpharmaceuticals, introduce improved possibilities to combat cancer.Further, the present invention discloses a method of synthesis of thesecompounds, and a modified form of a cytostatic pharmaceutical compound;doxorubicin.

DESCRIPTION OF THE INVENTION

It has been shown that the amount of lipoperoxides arise after theaction of doxorubicin (DXR) in the presence of docosahexaenoic acid andoxidants, in the human breast cancer cells (line MDA-MB-231). This mayendow tumour cells with metabolic characteristics that decrease theirpropensity to survive the effects of doxorubicin.

The present inventor has previously made available novel amides of theall-trans-retinoic acid or 13-cis-retinoic acid, arachidonic acid,docosahexaenoic acid and eicosapentaenoic acid or linolenic acid with2-aminoehtanol, alpha-L-serine, alpha-L-threonine, alpha-L-tyrosinecontaining phosphate groups (SE 9900941-7, filed on Mar. 16, 1999). Thepresent invention discloses the use of specific compounds, in particulartheir application for increase the pharmacological activity ofdoxorubicin.

These novel compounds contain hydrophobic residues of polyunsaturatedfatty acids, retinoic acid residues and a phosphate group, which has anegative charge. Thus, the interaction between molecules of novelcompounds and doxorubicin could be realised by hydrophobic interactionbetween fatty acid residues or retinoic acid residues and the planarchromophore of doxorubicin, as well as an electrostatic interactionbetween contrary charged functional groups both compounds.

On the one hand these binary complexes have all necessaries propertiesfor directed transport through the membrane of the cancer cells andresemble a “Trojan horse”. On the other hand, the dissociation of thesebinary complexes inside of the cancer cells releases “native”, positivecharged molecules of doxorubicin, with the result that favourableconditions for doxorubicin intercalation into DNA are created.

The following compounds 1 through 4, 1a through 4a, and 5 through 20form the basis of the invention. They have—in part—been disclosed in theSwedish patent application no. 9900941-7, filed on Mar. 16, 1999.

Retinoic Acid Derivatives:

1. N-(all-trans-retinoyl)-o-phospho-2-aminoethanol

1a. N-(13-cis-retinoyl)-o-phospho-2-aminoethanol

2. N-(all-trans-retinoyl)-o-phospho-L-serine

2a. N-(13-cis-retinoyl)-o-phospho-L-serine

3. N-(all-trans-retinoyl)-o-phospho-L-threonine

3a. N-(13 -cis-retinoyl)-o-phospho-L-threonine

4. N-(all-trans-retinoyl)-o-phospho-L-tyrosine

4a. N-(13-cis-retinoyl)-o-phospho-L-tyrosine

Arachidonic Acid Derivatives:

5. N-arachidonoyl-o-phospho-2-aminoethanol

6. N-arachidonoyl-o-phospho-L-serine

7. N-arachidonoyl-o-phospho-L-threonine

8. N-arachidonoyl-o-phospho-L-tyrosine

Docosahexaenoic Acid Derivatives:

9. N-docosahexaenoyl-o-phospho-2-aminoethanol

10. N-docosahexaenoyl-o-phospho-L-serine

11. N-docosahexaenoyl-o-phospho-L-threonine

12. N-docosahexaenoyl-o-phospho-L-tyrosine

Eicosapentaenoic Acid Derivatives:

13. N-eicosapentaenoyl-o-phospho-2-aminoethanol

14. N-eicosapentaenoyl-o-phospho-L-serine

15. N-eicosapentaenoyl-o-phospho-L-threonine

16. N-eicosapentaenoyl-o-phospho-L-tyrosine

Linoleic Acid Derivatives:

17. N-linolenoyl-o-phospho-2-aminoethanol

18. N-linolenoyl-o-phospho-L-serine

19. N-linolenoyl-o-phospho-L-threonine

20. N-linolenoyl-o-phospho-L-tyrosine

In the following description and examples, the above compounds arereferred to as C1 through C4, C1a-C4a, and C5-C20.

A study of the anti-tumour effect of complexes between doxorubicin (DXR)and any one of the above compounds C1-C4, C1a-C4a, and C5-C20, ascompared with DXR alone, was carried out using mice with EAC (Ehrlichascites carcinoma). The extent of inhibition of EAC growth in mice,achieved by the tested compounds, compared to DXR, was used forevaluation of the anti-tumour activity of each tested DXR/compoundcomplex.

It has been experimentally shown, that the complex DXR with any compoundalone (C1-C4, C1a-C4a, and C5-C20) did not display an anti-tumoureffect. In particular, in the DXR/C4 complex, the compound C4 cancelledthe anti-tumour action of DXR and even exhibited some (insignificantlysmall) stimulating influence on EAC growth in mice. In the DXR/C5complex, compound C5 cancelled the anti-tumour action of DXR.

The present inventor has however shown that complexes of DXR and C4 orC4a, together with C5; DXR/C4 (C4a) with C9; DXR/C4 (C4a) with C13; andDXR/C4 (C4a) with C17 display anti-tumour effects.

The attached series of experimental results, support this finding. Thefollowing doses were used:

DXR—3.5 mg/kg of body weight

C4—8.15 mg/kg of body weight

At the molar ratios C4:C5 equal to 1:3; 1:2.9; 1:2.8; 1:2.7 and 1:2.6,the EAC growth inhibition was 45.0%; 43.6%; 46.0%; 48.2%; 50.4%,respectively.

At the molar ratios C4:C5: equal to 1:2.5; 1:2.4; 1:2.3 and 1:2.2, theEAC growth inhibition was 48.7%; 51.5%; 53.4% and 57.0%, respectively.

At the molar ratios C4:C5: equal to 1:2.1; 1:2; 1:1.9 and 1:1.8, the EACgrowth inhibition was 58.0%; 59.5%; 62.0% and 65.2%, respectively.

At the molar ratios C4:C5 equal to 1:1.7; 1:1.6; 1:1.5 and 1:1.4, theEAC growth inhibition was 67.1%; 67.9%; 69.1% and 70.0%, respectively.

At the molar ratios C4:C5 equal to 1:13; 1:1.2; 1:1.1 and 1:1, the EACgrowth inhibition was 69.4%; 66.7%; 62.5% and 65.2%, respectively.

It should be noted that, at the molar ratios C4:C5 equal to 1:1.7;1:1.6; 1:1.5; 1:1.4 and 1:1.3, the EAC growth inhibition with referenceto the DXR group (positive control) was 38.8%; 40.3%; 42.6%; 44.2% and41.1%, respectively.

DXR—3.5 mg/kg of body weight

CS—6.4 mg/kg of body weight

At the molar ratios C4:C5 equal to 1:1; 1.1:1; 1.2:1; 1.3:1 and 1.4:1,the EAC growth inhibition was 65.2%; 61.6%; 66.2%; 69.3% and 69.8%,respectively.

At the molar ratios C4:C5 equal to 1.5:1; 1.6:1; 1.7:1 and 1.8:1, theEAC growth inhibition was 74.9%; 78.1%; 74.1% and 66.4%, respectively.

At the molar ratios C4:C5 equal to 1.9:1; 2:1; 2.1:1 and 2.2:1, the EACgrowth inhibition was 65.1%; 61.8%; 63.2% and 58.0%, respectively.

At the molar ratios C4:C5 equal to 2.3:1; 2.4:1; 2.5:1 and 2.6:1, theEAC growth inhibition was 55.5%; 58.7%; 56.7% and 57.6%, respectively.

At the molar ratios C4:C5 equal to 2.7:1; 2.8:1; 2.9:1 and 3:1, the EACgrowth inhibition was 56.6%; 55.0%; 50.4% and 45.3%, respectively.

It should be noted that, at the molar ratios C4:C5 equal to 1.3:1;1.4:1; 1.5:1; 1.6:1 and 1.7:1, the EAC growth inhibition with referenceto the DXR group (positive control) was 43.2%; 44.1%; 51.4%; 57.7% and49.8%, respectively.

In particular, some of the experiments for testing the anti-tumoureffects of DXR/C4+C9 complexes, DXR/C4+C13 complexes, DXR/C4+C17complexes, DXR/C4a+C5 complexes indicate this.

DXR/C4+C9 Complexes:

DXR—3.5 mg/kg of body weight, and C4—8.15 mg/kg of body weight

At the molar ratios C4:C9 equal to 1:1; 1:1.4; 1:1.8 and 1:2.3, the EACgrowth inhibition was 61.8%; 67.3%; 62.5% and 49.7%, respectively.

At the molar ratio C4:C9 equal to 1:1.4, the EAC growth inhibition withreference to the DXR group (positive control) was 49.3%.

DXR/C4+C13 Complexes:

DXR—3.5 mg/kg of body weight, and C4—8.15 mg/kg of body weight.

At the molar ratios C4:C13 equal to 1:1.3; 1:1.6; 1:2 and 1:2.5, the EACgrowth inhibition was 67.0%; 64.6%; 54.2% and 45.1%, respectively.

At the molar ratios C4:C13 equal to 1:1.3 and 1:1.6, the EAC growthinhibition with reference to the DXR group (positive control) was 47.6%and 43.8%, respectively.

DXR/C4+C17 Complexes:

DXR—3.5 mg/kg of body weight, and C17—6,0 mg/kg of body weight.

At the molar ratios C4:C17 equal to 1:1; 1.4:1; 2:1 and 2.7:1, the EACgrowth inhibition was 62.9%; 67.4%; 60.3% and 51.8%, respectively.

At the molar ratio C4:C17 equal to 1.4:1, the EAC growth inhibition withreference to the DXR group (positive control) was 45.0%.

DXR/C4a+C5 Complexes:

DXR—3.5 mg/kg of body weight, and C5—6,4 mg/kg of body weight.

At the molar ratios C4a:C5 equal to 1.2:1; 1.6:1; 1.9:1 and 2.5:1, theEAC growth inhibition was 64.4%; 72.4%; 62.2% and 52.5%, respectively.

At the molar ratio C4a:C5 equal to 1.6:1, the EAC growth inhibition withreference to the DXR group (positive control) was 49.4%.

The present inventor has shown that DXR/(C4+C4a)+C5 complexes;DXR/(C4+C4a)+(C5+C9+C13) complexes; DXR/(C4+C4a)+(C5+C9+C13+C17)complexes display an anti-tumour effect. The experiments for testing theanti-tumour effect support this finding.

DXR/(C4+C4a)+C5 complexes: DXR—3.5 mg/kg of body weight

C5—6.4 mg/kg of body weight

At the molar ratios (C4+C4a):C5 equal to 1.2:1; 1.6:1; 1.9:1 and 2.5:1EAC growth inhibition was 63.1%; 71.6%; 61.0% and 47.3%, respectively.

At the molar ratios (C4+C4a):C5 equal to 1.6:1 EAC growth inhibitionwith reference to DXR group (positive control) was 53.1%.

DXR/(C4+C4a)+(C5+C9+C13) complexes: DXR—3.5 mg/kg of body weight

(C4+C4a)—8.15 mg/kg of body weight

At the molar ratios (C4+C4a):(C5+C9+C13) equal to 1:1; 1:1.4; 1:1.8 and1:2.3 EAC growth inhibition was 60.1%; 68.3%; 61.3% and 47.2%,respectively.

At the molar ratios (C4+C4a):(C5+C9+C13) equal to 1:1.4 EAC growthinhibition with reference to DXR group (positive control) was 49.8%.

DXR/(C4+C4a)+(C5+C9+C13+C17) complexes: DXR—3.5 mg/kg of body weight

(C4+C4a)—8.15 mg/kg of body weight

At the molar ratios (C4+C4a):(C5+C9+C13+C17) equal to 1:1.3; 1:1.6; 1:2and 1:2.5 EAC growth inhibition was 68.0%; 69.7%; 56.3% and 43.5%,respectively.

At the molar ratios (C4+C4a):(C5+C9+C13+C17) equal to 1:1.3 and 1:1.6EAC growth inhibition with reference to DXR group (positive control) was45.3% and 48.1% respectively.

Investigations of the anti-tumour effects of DXR/C4+C5 complexes,DXR/C4+C9 complexes, DXR/C4+C13 complexes, DXR/C4+C17 complexes,DXR/C4a+C5 complexes allow the following conclusion:

At the molar ratios C4(or C4a):C5 (or C9 or C13 or C17) from 1:3 to 1:1and from 1:1 to 3:1, the anti-tumour action complexes exceed the effectof DXR alone. The results show significant effects for complexes withinthe intervals 1:2-1:1 and 1:1-2:1, with improved effects correspondingto the more narrow intervals 1:1.7-1:1.3 and 1.3:1-1.7:1.

The investigation of anti-tumour effects of DXR/(C4+C4a)+C5 complexes,DXR/(C4+C4a)+(C5+C9+C13) complexes, DXR/(C4+C4a)+(C5+C9+C13+C17)complexes confirm this conclusion. Moreover, it is true for complexes,in which from two to six compounds (4, 4a, 5, 9, 13, 17) participate ininteractions with DXR.

These results were obtained at such concentrations of novel compounds(C4, C4a, C5, C9, C13, C17) which are considerably above their criticalmicelle concentrations.

A water-soluble aromatic compound, such as doxorubicin could be in theform of micelle in water solutions. The present inventor hassurprisingly found, that doxorubicin in a concentration interval of 1-2mg/ml forms mixed micelles with the novel compounds (C4, C4a, C5, C9,C13, C17) more effectively. Further, the anti-tumour activity of thesemixed micelles is significantly higher then for doxorubicin alone in thesame concentrations.

Thus, mixed micelles consisting of an amide of the all-trans-retinoicacid or/and an amide of the 13-cis-retinoic acid withO-phospho-L-tyrosine (C4 or C4a; C4+C4a), amides of polyunsaturatedacids with O-phosphorylethanolamine (C5 or C9 or C13 or C17; C5+C9;C5+C13; C5+C17; C9+C13; C9+C17; C13+C17; C5+C9+C13; C5+C9+C17;C5+C13+C17; C9+C13+C17; C5+C9+C13+C17) and doxorubicin, displayanti-tumour activity.

Thus, mixed micelles consisting of amide of the all-trans-retinoic acidor 13-cis-retinoic acid with O-phospho-L-tyrosine (C4 or C4a), amidepolyunsaturated acid with O-phosphorylethanolamine (C5 or C9 or C13 orC17) and doxorubicin, display anti-tumour activity.

The inventor has previously described a universal method of synthesisamides of retinoic acids and polyunsaturated acids with hydroxyaminoacids and ethanolamine containing phosphate groups. In the presentapplication, the inventor discloses a method of synthesis of theN-acyl-O-phospho-2-aminoethanol and the N-retinoyl-O-phospho-L-tyrosine.

That method has the following advantages in comparison with previous one

the synthesis is performed in one step

the yield is high, reaching 90%,

simplified and time-saving synthesis

the final product can be purified without chromatography

EXAMPLES

Materials and Methods:

The study of the anti-tumour effects of complexes between doxorubicinand compound 4 (C4) or compound 4a (C4a) and compound 5 (CS) or compound9 (C9) or compound 13 (C13) or compound 17 (C17) as compared todoxorubicin (DXR) was carried out in mice with Ehrlich ascites carcinoma(EAC). Random-bred albino mice of line ICR, male and female, from ownrearing were used in the study. The room for animal housing was providedwith filtered air at 15 changes per hour, a temperature of 19-21° C.,relative humidity of 50-60% and regulated light day of 12 hours withchange of light and darkness at 6 a.m. and 6 p.m. The mice were kept intransparent polycarbonate cages with a floor area of 600 cm² containingsoftwood sawdust bedding. The animals had free access to bottles withdomestic quality drinking tap water and to standard feeding for growinganimals ad libidum. The mice aged 2-2.5 months and having a weight of19-22 g were randomly distributed in the control and test groups,standardised by mean weights. The control and test groups included 10and 7 animals correspondingly. The mice of each group were identified byspecific ear marks. The cages with animals of each group were identifiedby cage cards marked with the study date, group number, number and sexof the animals.

The strain of EAC was propagated in ICR mice by intraperitonealinoculation of 0.2 ml native ascites fluid or 0.5 ml ascites fluiddiluted by saline to 1:1 from one or two animals with intraperitoneallygrowing EAC on day 7. Ascites fluids contained more 98% of viable tumourcells accordig to trypan blue exclusion test. For the each study thesuspension of EAC cells in sterile saline in concentration of 10⁷ viabletumour cells/ml was prepared in aseptic conditions from ascites fluid ofmouse bearing of EAC on day 7.

The mice of each control and experimental groups were inoculated byintraperitoneal injection of 2×10⁶ EAC cells in volume of 0.2 ml at day0. Intravenous injections for the study were carried out in lateral tailvein of mice once every other day, three times (day 2, 4 and 6)following inoculation of EAC cells. The mice of control group wereinjected by vehicle in volume of 2.5 ml/kg of body weight (50 μl tomouse of 20 g body weight) intravenously (negative control). Other groupof animals (positive control) received water solution of DXR (inconcentration of 1.4 mg/ml) in the dose of 3.5 mg/kg of body weight andin volume of 2.5 ml/kg of body weight (70 μg of DXR in 50 μl to mouse of20 g body weight) intravenously. The mice of test groups were injectedintravenously by complexes of DXR/C4+C5 (two series of experiments);complexes of DXR/C4+C9 (some experiments); complexes of DXR/C4+C13 (someexperiments); complexes of DXR/C4+C17 (some experiments); complexesof-DXR/C4a+C5 (some experiments). The mice of test groups received theDXR (in complexes) in the dose of 3.5 mg/kg of body weight and in volumeof 2.5 ml/kg of body weight (corresponding to 70 μg of DXR in 50 μl tomouse of 20 g body weight); concentration of DXR 1,4 mg/ml. One seriesof experiments was designated to study the anti-tumour effect ofcomplexes of DXR/C4+C5 at constant content of C4 (8.15 mg/kg of bodyweight) but at different content of C5 with molar ratio C4:C5, varyingfrom 1:3 to 1:1. Other series of experiments was designated to study theanti-tumour effect of complexes of DXR/C4+C5 at constant content of C5(6.4 mg/kg of body weight) but at different content of C4 with molarratio C4:C5, varying from 1:1 to 3:1. Solutions of complexes anddoxorubicin were injected strictly intravenously as occasional partialsubcutaneous injection can lead to pain reaction of animal, localirritant damage, or ulceration and necrosis at times.

The mice were killed by cervical dislocation two days later after thefinal treatment with the test complexes on day 8. Ascites fluids wereremoved, collected, their volumes were recorded, and abdominal cavitieswere washed by saline 6-7 times and both fluids were pooled. Aftercentrifugation at 1000 r.p.m. for 10 min volumes of tumour cell pelletwere also recorded. Number of viable tumour cells was counted byhemocetometer. Means and standard errors for each groups werecalculated. Comparison of tumour cell number in control and test groupswas carried out using Student's t-test. The influence of testedpreparations on EAC growth inhibition was evaluated by the followingformula:$\quad {{inhibition},{\% = {\frac{{Control} - {Test}}{Control} \times 100}}}$

The extent of inhibition of EAC growth in mice, affected by the testpreparations compared to DXR, was used for evaluation of anti-tumouractivity of tested complexes.

Example 1

Anti-tumour Effect of DXR/C4 Complex

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg of body weight) once everyother day, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of test group received the complex of DXR/C4 (DXR—3.5 mg/kg;C4—22.5 mg/kg). Two days later after the final treatment with testcomplex mice were killed by cervical dislocation on day 8. Tumour cellnumber was counted and the extent of inhibition of EAC growth in micewas evaluated.

Mice of control group had (680.0±59.1)×10⁶ EAC cells in abdominalcavity. In the group receiving DXR alone, the number of tumour cells was(510.9±52.9)×10⁶ and EAC growth inhibition 24,9%, p<0.05. In mice in thetest group receiving the DXR/C4 complex, the number of tumour cells was(787.2±141.8)×10⁶, p>0.05.

Thus, the DXR/C4 complex did not display an anti-tumour action.Moreover, compound 11 canceled the anti-tumour action of DXR and exertedinsignificantly some stimulating influence on EAC growth in mice withreference to negative control.

Example 2

Anti-tumour Effect of DXR/C5 Complex

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg of body weight) once everyother day, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of test group received the DXR/C5 complex (DXR—3.5 mg/kg;C5—12 mg/kg). Two days later after the final treatment with test complexmice were killed by cervical dislocation on day 8. Tumour cell numberwas counted and the extent of inhibition of EAC growth in mice wasevaluated.

Mice of control group had (680.0±59.1)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(510.9±52.9)×10⁶ and EAC growth inhibition of 24,9%, p<0.05. In mice oftest group with DXR/C5 complex the number of tumour cells was(635.6±122.2)×10⁶, p>0.05.

Thus, the DXR/C5 complex did not display an anti-tumour action.Moreover, C5 canceled the anti-tumour action of DXR.

Series of experiments for testing of anti-tumour effect of DXR/C4+C5complexes. DXR—3.5 mg/kg of body weight. C4—8.15 mg/kg of body weight.C4:C5 molar ratio varied from 1:3 to 1:1.

Example 3

The anti-tumour Effect of DXR/C4+C5 complexes, the C4:C5 Molar RatiosBeing Equal to 1:3; 1:2,9; 1:2.8; 1:2.7 and 1:2.6

ICR-mice weighing 20-22 g were inoculated intraperitoneally with 2×10⁶viable EAC cells. Starting two days later (day 2), the mice wereinjected intravenously (with a volume corresponding to 2.5 ml/kg bodyweight) once every other day, three times (day 2, 4 and 6). Mice ofcontrol group received only vehicle. In the group of DXR mice receivedDXR alone in the dose of 3.5 mg/kg. Mice of first test group receivedthe DXR/C4+C5 complex (C4:C15 molar ratio was equal to 1:3). Mice ofsecond test group received the DXR/C4+C5 complex (C4:C5 molar ratio wasequal to 1:2.9). Mice of third test group received the DXR/C4+C5 complex(C4+C5 molar ratio was equal to 1:2.8). Mice of fourth test groupreceived the DXR/C4+C5 complex (C4:C5 molar ratio was equal to 1:2.7).Mice of fifth test group received the DXR/C4+C5 complex (C4:C5 molarratio was equal to 1:2.6). Two days later after the final treatment withthe test complexes mice were killed by cervical dislocation on day 8.Tumour cell number was counted and the extent of inhibition of EACgrowth in mice was evaluated.

Mice of control group had (657.5±72.6)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(407.4±75.9)×10⁶ and EAC growth inhibition of 38.0%, p<0.05. In mice offirst test group the number of tumour cells was (361.3±98.5)×10⁶ and EACgrowth inhibition of 45.0%, p<0.05. In mice of second test group thenumber of tumour cells was (370.6±94.9)×10⁶ and EAC growth inhibition of43.6%, p<0.05. In mice of third test group the number of tumour cellswas (354.9±101.2)×10⁶ and EAC growth inhibition of 46.0%, p<0.05. Inmice of fourth test group the number of tumour cells was(340.8±104.7)×10⁶ and EAC growth inhibition of 48.2%, p<0.05. In mice offifth test group the number of tumour cells was (326.1±93.3)×10⁶ and EACgrowth inhibition of 50.4%, p<0.05.

Thus, the anti-tumour activity of DXR/C4+C5 complexes at various C4:C5molar ratios from 1:3.0 to 1:2.6 exceeds the effect of DXR alone.

Example 4

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 1:2.5; 1:2.4; 1:2.3 and 1:2.2

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 1:2.5). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 1:2.4). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 1:2.3). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 1:2.2). Two days later after the finaltreatrnent with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (935.1±107.8)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(548.5±81.7)×10⁶ and EAC growth inhibition of 41.3%, p<0.02. In mice offirst test group the number of tumour cells was (479.4±103.8)×10⁶ andEAC growth inhibition of 48.7%, p<0.01. In mice of second test group thenumber of tumour cells was (453.7±125.1)×10⁶ and EAC growth inhibitionof 51.5%, p<0.02. In mice of third test group the number of tumour cellswas (435.5±99.8)×10⁶ and EAC growth inhibition of 53.4%, p<0.01. In miceof fourth test group the number of tumour cells was (402.0±116.9)×10⁶and EAC growth inhibition of 57.0%, p<0.01.

Thus, anti-tumour activity of DXR/C4+C5 complexes at various molar C4:C5ratios from 1:2.5 to 1:2.2 exceeds the effect of DXR alone.

Example 5

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 1:2.1; 1:2; 1:1.9 and 1:1.8

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 1:2.1). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 1:2). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 1:1.9). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 1:1.8). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (862.4±125.0)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(455.3±131.3)×10⁶ and EAC growth inhibition of 47.2%, p<0.05. In mice offirst test group the number of tumour cells was (361.8±79.4)×10⁶ and EACgrowth inhibition of 58.0%, p<0.01. In mice of second test group thenumber of tumour cells was (349.3±92.2)×10⁶ and EAC growth inhibition of59.5%, p<0.01. In mice of third test group the number of tumour cellswas (327.5±108.6)×10⁶ and EAC growth inhibition of 62.0%, p<0.01. Inmice of fourth test group the number of tumour cells was(300.2±84.7)×10⁶ and EAC growth inhibition of 65.2%, p<0.01.

Thus, anti-tumour activity of DXR/C4+C5 complexes at various C4:C5 molarratios from 1:2.1 to 1:1.8 exceeds the effect of DXR alone.

Example 6

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 1:1.7; 1:1.6; 1:1.5 and 1:1.4

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg, Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 1:1.7). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 1:1.6). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 1:1.5). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 1:1.4). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (779.1±76.7)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(419.0±53.4)×10⁶ and EAC growth inhibition of 46.2%, p<0.002. In mice offirst test group the number of tumour cells was (256.3±54.1)×10⁶ and EACgrowth inhibition of 67.1%, p<0.001, and with reference to DXR group(positive control) EAC growth inhibition of 38.8%, p<0.05. In mice ofsecond test group the number of tumour cells was (250.1±57.7)×10⁶ andEAC growth inhibition of 67.9%, p<0.001, and with reference to DXR group(positive control) EAC growth inhibition of 40.3%, p<0.05. In mice ofthird test group the number of tumour cells was (240.7±61.8)×10⁶ and EACgrowth inhibition of 69.1%, p<0.001, and with reference to DXR group(positive control) EAC growth inhibition of 42.6%, p<0.05. In mice offourth test group the number of tumour cells was (233.7±50.8)×10⁶ andEAC growth inhibition of 70.0%, p<0.001, and with reference to DXR group(positive control) EAC growth inhibition of 44.2%, p<0.05.

Thus, the anti-tumour activity of DXR/C4+C5 complexes at various C4:C5molar ratios from 1:1.7 to 1:1.4 exceeds the effect of DXR alone.

Example 7

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 1:1.3; 1:1.2; 1:1.1 and 1:1

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 1:1.3). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 1:1.2). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 1:1.1). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 1:1). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (793.4±72.8)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(411.9±57.1)×10⁶ and EAC growth inhibition of 48.1%, p<0.02. In mice offirst test group the number of tumour cells was (242.6±54.3)×10⁶ and EACgrowth inhibition of 69.4%, p<0.001, and with reference to DXR group(positive control) EAC growth inhibition of 41.1%, p<0.05. In mice ofsecond test group the number of tumour cells was (264.1±87.6)×10⁶ andEAC growth inhibition of 66.7%, p<0.001. In mice of third test group thenumber of tumour cells was (297.7±94.3)×10⁶ and EAC growth inhibition of62.5%, p<0.001. In mice of fourth test group the number of tumour cellswas (276.1±76.5)×10⁶ and EAC growth inhibition of 65.2%, p<0.001.

Thus, anti-tumour activity of DXR/C4+C5 complexes at various C4:C5 molarratios from 1:1.3 to 1:1 exceeds the effect of DXR alone.

Series of experiments for testing of anti-tumour effect of DXR/C4+C5complexes. DXR—3.5 mg/kg of body weight. C5—6.4 mg/kg of body weight.C4:C5 molar ratio varied from 1:1 to 3:1.

Example 8

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 1:1; 1.1:1; 1.2:1; 1.3:1 and 1.4:1

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 1:1). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 1.1:1). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 1.2:1). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 1.3:1). Mice of fifth test groupreceived the DXR/C4+C5 complex (C4:C5 molar ratio was equal to 1.4:1).Two days later after the final treatment with the test complexes micewere killed by cervical dislocation on day 8. Tumour cell number wascounted and the extent of inhibition of EAC growth in mice wasevaluated.

Mice of control group had (742.2±66.2)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(400.3±63.5)×10⁶ and EAC growth inhibition of 46.1%, p<0.001. In mice offirst test group the number of tumour cells was (258.3±40.4)×10⁶ and EACgrowth inhibition of 65.2%, p<0.001. In mice of second test group thenumber of tumour cells was (284.8±97.5)×10⁶ and EAC growth inhibition of61.6%, p<0.002. In mice of third test group the number of tumour cellswas (251.2±103.6)×10⁶ and EAC growth inhibition of 66.2%, p<0.001. Inmice of fourth test group the number of tumour cells was(227.5±43.3)×10⁶ and EAC growth inhibition of 69.3%, p<0.001, and withreference to DXR group (positive control) EAC growth inhibition of43.2%, p<0.05. In mice of fifth test group the number of tumour cellswas (223.8±43.2)×10⁶ and EAC growth inhibition of 69.8%, p<0.001, andwith reference to DXR group (positive control) EAC growth inhibition of44.1%, p<0.05.

Thus, anti-tumour activity of DXR/C4+C5 complexes at various C4:C5 molarratios from 1:1 to 1.4:1 exceeds the effect of DXR alone.

Example 9

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 1.5:1; 1.6:1; 1.7:1 and 1.8:1

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 1.5:1). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 1.6:1). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 1.7:1). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 1.8:1). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (953.5±167.3)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(492.1±85.3)×10⁶ and EAC growth inhibition of 48.4%, p<0.05. In mice offirst test group the number of tumour cells was (239.3±93.5)×10⁶ and EACgrowth inhibition of 74.9%, p<0.002, and with reference to DXR group(positive control) EAC growth inhibition of 51.4%, p<0.05. In mice ofsecond test group the number of tumour cells was (208.4±89.0)×10⁶ andEAC growth inhibition of 78.1%, p<0.002, and with reference to DXR group(positive control) EAC growth inhibition of 57.7%, p<0.05. In mice ofthird test group the number of tumour cells was (247.1±76.8)×10⁶ and EACgrowth inhibition of 74.1%, p<0.002, and with reference to DXR group(positive control) EAC growth inhibition of 49.8%, p<0.05. In mice offourth test group the number of tumour cells was (320.4±120.6)×10⁶ andEAC growth inhibition of 66.4%, p<0.01.

Thus, the anti-tumour activity of DXR/C4+C5 complexes at various C4:C5molar ratios from 1.5:1 to 1.8:1 exceeds the effect of DXR alone.

Example 10

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 1.9:1; 2:1; 2.1:1 and 2.2:1

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg of body weight) once everyother day, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 1.9:1). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 2:1). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 2.1:1). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 2.2:1). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (924.6±145.2)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(481.0±105.3)×10⁶ and EAC growth inhibition of 48.0%, p<0.01. In mice offirst test group the number of tumour cells was (322.8±93.5)×10⁶ and EACgrowth inhibition of 65.1%, p<0.01. In mice of second test group thenumber of tumour cells was (353.2±74.4)×10⁶ and EAC growth inhibition of61.8%, p<0.01. In mice of third test group the number of tumour cellswas (340.3±86.8)×10⁶ and EAC growth inhibition of 63.2%, p<0.01. In miceof fourth test group the number of tumour cells was (388.3±91.6)×10⁶ andEAC growth inhibition of 58.0%, p<0.01.

Thus, the anti-tumour activity of DXR/C4+C5 complexes at various C4:C5molar ratios from 1.9:1 to 2.2:1 exceeds the effect of DXR alone.

Example 11

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 2.3:1; 2.4:1; 2.5:1 and 2.6:1

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg of body weight) once everyother day, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 2.3:1). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 2.4:1). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 2.5:1). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 2.6:1). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (857.6±113.9)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(525.2±104.5)×10⁶ and EAC growth inhibition of 38.8%, p<0.05. In mice offirst test group the number of tumour cells was (381.4±64.8)×10⁶ and EACgrowth inhibition of 55.5%, p<0.01. In mice of second test group thenumber of tumour cells was (354.2±78.1)×10⁶ and EAC growth inhibition of58.7%, p<0.01. In mice of third test group the number of tumour cellswas (371.4±136.5)×10⁶ and EAC growth inhibition of 56.7%, p<0.02. Inmice of fourth test group the number of tumour cells was(363.7±158.4)×10⁶, inhibition of EAC growth was 57.6%, p<0.05.

Thus, the anti-tumour activity of DXR/C4+C5 complexes at various C4:C5molar ratios from 2.3:1 to 2.6:1 exceeds the effect of DXR alone.

Example 12

The Anti-tumour Effect of DXR/C4+C5 Complexes, the C4:C5 Molar RatiosBeing Equal to 2,7:1; 2.8:1; 2,9:1 and 3:1

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg of body weight) once everyother day, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C5 complex (C4:C5molar ratio was equal to 2.7:1). Mice of second test group received theDXR/C4+C5 complex (C4:C5 molar ratio was equal to 2.8:1). Mice of thirdtest group received the DXR/C4+C5 complex (C4:C5 molar ratio was equalto 2.9:1). Mice of fourth test group received the DXR/C4+C5 complex(C4:C5 molar ratio was equal to 3:1). Two days later after the finaltreatment with test complexes mice were killed by cervical dislocationon day 8. Tumour cell number was counted and the extent of inhibition ofEAC growth in mice was evaluated.

Mice of control group had (721.3±76.4)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(503.8±64.5)×10⁶ and EAC growth inhibition of 30.2%, p<0.05. In mice offirst test group the number of tumour cells was (312.7±79.6)×10⁶ and EACgrowth inhibition of 56.6%, p<0.002. In mice of second test group thenumber of tumour cells was (324.5±72.8)×10⁶ and EAC growth inhibition of55.0%, p<0.002. In mice of third test group the number of tumor cellswas (357.9±75.3)×10⁶ and EAC growth inhibition of 50.4%, p<0.01. In miceof fourth test group the number of tumour cells was (394.6±67.7)×10⁶ andEAC growth inhibition of 45.3%, p<0.01.

Thus, anti-tumour activity of DXR/C4+C5 complexes at various C4:C5 molarratios from 2.7:1 to 3:1 exceeds the effect of DXR alone.

Example 13

The Anti-tumour Effect of DXR/C4+C9 Complexes, the C4:C9 Molar RatiosBeing Equal to 1:1; 1:1.4; 1:1.8 and 1:2.3. DXR—3.5 mg/kg of BodyWeight. C4—8.15 mg/kg of Body Weight

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C9 complex (C4:C9molar ratio was equal to 1:1). Mice of second test group received theDXR/C4+C9 complex (C4:C9 molar ratio was equal to 1:1.4). Mice of thirdtest group received the DXR/C4+C9 complex (C4:C9 molar ratio was equalto 1:1.8). Mice of fourth test group received the DXR/C4+C9 complex(C4:C9 molar ratio was equal to 1:2.3). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (696.7±68.2)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(449.4±77.1)×10⁶ and EAC growth inhibition of 35.5%, p<0.05. In mice offirst test group the number of tumour cells was (266.1±97.9)×10⁶ and EACgrowth inhibition of 61.8%, p<0.01. In mice of second test group thenumber of tumour cells was (227.8±69.1)×10⁶ and EAC growth inhibition of67.3%, p<0.001, and with reference to DXR group (positive control) EACgrowth inhibition of 49.3%, p<0.05. In mice of third test group thenumber of tumour cells was (261.0±101.2)×10⁶ and EAC growth inhibitionof 62.5%, p<0.01. In mice of fourth test group the number of tumourcells was (350.4±103.7)×10⁶ and EAC growth inhibition of 49.7%, p<0.02.

Thus, the anti-tumour activity of DXR/C4+C9 complexes at C4:C9 molarratios 1:1; 1:1.4; 1:1.8 and 1:2.3 exceeds the effect of DXR alone.

Example 14

Anti-tumour Effect of DXR/C4+C13 Complexes. C4:C13 Molar Ratios wereEqual to 1:1.3; 1:1.6; 1:2 and 1:2.5. DXR—3.5 mg/kg of Body Weight.C4—8.15 mg/kg of Body Weight

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C13 complex (C4:C13molar ratio was equal to 1:1.3). Mice of second test group received theDXR/C4+C13 complex (C4:C13 molar ratio was equal to 1:1.6). Mice ofthird test group received the DXR/C4+C13 complex (C4:C13 molar ratio wasequal to 1:2). Mice of fourth test group received the DXR/C4+C13 complex(C4:C13 molar ratio was equal to 1:2.5). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (645.8±62.5)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(406.7±60.1)×10⁶ and EAC growth inhibition of 37.0%, p<0.05. In mice offirst test group the number of tumour cells was (213.1±53.0)×10⁶ and EACgrowth inhibition of 67.0%, p<0.001, and with reference to DXR group(positive control) EAC growth inhibition of 47.6%, p<0.05. In mice ofsecond test group the number of tumour cells was (228.6±57.0)×10⁶ andEAC growth inhibition of 64.6%, p<0.001 and with reference to DXR group(positive control) EAC growth inhibition of 43.8%, p<0.05. In mice ofthird test group the number of tumour cells was (295.8±94.0)×10⁶ and EACgrowth inhibition of 54.2%, p<0.01. In mice of fourth test group thenumber of tumour cells was (354.5±75.6)×10⁶ and EAC growth inhibition of45.1%, p<0.01.

Thus, anti-tumour activity of DXR/C4+C13 complexes at C4:C13 molarratios 1:1.3; 1:1.6; 1:2 and 1:2.5 exceeds the effect of DXR alone.

Example 15

Anti-tumour Effect of DXR/C4+C17 Complexes. C4:C17 Molar Ratios wereEqual to 1:1; 1.4:1; 2:1 and 2.7:1. DXR—3.5 mg/kg of Body Weight.C17—6.0 mg/kg of Body Weight

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4+C17 complex (C4:C17molar ratio was equal to 1:1). Mice of second test group received theDXR/C4+C17 complex (C4:C17 molar ratio was equal to 1.4:1). Mice ofthird test group received the DXR/C4+C17 complex (C4:C17 molar ratio wasequal to 2:1). Mice of fourth test group received the DXR/C4+C17 complex(C4:C17 molar ratio was equal to 2.7:1). Two days later after the finaltreatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (834.9±71.1)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(495.1±68.0)×10⁶ and EAC growth inhibition of 40.7%, p<0.01. In mice offirst test group the number of tumour cells was (309.7±71.7)×10⁶ and EACgrowth inhibition of 62.9%, p<0.001. In mice of second test group thenumber of tumour cells was (272.2±68.5)×10⁶ and EAC growth inhibition of67.4%, p<0.001, and with reference to DXR group (positive control) EACgrowth inhibition of 45.0%, p<0.05. In mice of third test group thenumber of tumour cells was (331.5±74.8)×10⁶ and EAC growth inhibition of60.3%, p<0.001. In mice of fourth test group the number of tumour cellswas (402.4±83.9)×10⁶ and EAC growth inhibition of 51.8%, p<0.002.

Thus, the anti-tumour activity of DXR/C4+C17 complexes at C4:C17 molarratios 1:1; 1.4:1; 2:1 and 2.7:1 exceeds the effect of DXR alone.

Example 16

Anti-tumour Effect of DXR/C4a+C5 Complexes. C4a:C5 Molar Ratios wereEqual to 1.2:1; 1.6:1; 1.9:1 and 2.5:1. DXR—3.5 mg/kg of Body Weight.C5—6.4 mg/kg of Body Weight

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/C4a+C5 complex (C4a:C5molar ratio was equal to 1.2:1). Mice of second test group received theDXR/C4a+C5 complex (C4a:C5 molar ratio was equal to 1.6:1). Mice ofthird test group received the DXR/C4a+C5 complex (C4a:C5 molar ratio wasequal to 1.9:1). Mice of fourth test group received the DXR/C4a+C5complex (C4a:C5 molar ratio was equal to 2.5:1). Two days later afterthe final treatment with the test complexes mice were killed by cervicaldislocation on day 8. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (811.3±73.8)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(442.2±51.5)×10⁶ and EAC growth inhibition of 45.5%, p<0.001. In mice offirst test group the number of tumour cells was (288.8±89.6)×10⁶ and EACgrowth inhibition of 64.4%, p<0.001. In mice of second test group thenumber of tumour cells was (223.9±68.8)×10⁶ and EAC growth inhibition of72.4%, p<0.001, and with reference to DXR group (positive control) EACgrowth inhibition of 49.4%, p<0.05. In mice of third test group thenumber of tumour cells was (306.7±91.3)×10⁶ and EAC growth inhibition of62.2%, p<0.001. In mice of fourth test group the number of tumour cellswas (385.4±94.7)×10⁶ and EAC growth inhibition of 52.5%, p<0.01.

Thus, anti-tumour activity of DXR/C4a+C5 complexes at C4a:C5 molarratios 1.2:1; 1.6:1; 1.9:1 and 2.5:1 exceeds the effect of DXR alone.

Example 17

Synthesis of the N-Acyl-O-Phospho-2-Aminoethanol

Were Acyl is:

cis-5,8,11,14-eicosatetraenoyl (arachidonoyl) (5)

cis-4,7,10,13,16,19-docosahexaenoyl (9)

cis-5,8,11,14,17-eicosapentaenoyl (13)

cis-9,12,15-octadecatrienoyl (linolenoyl) (17)

Polyunsaturated acid (1 mmol) and triethylamine (142 μl, 1,02 mmol) weredissolved in 2 ml of dry tetrahydrofuran, then dry acetonitrile (4 ml)was added, the mixture chilled to −15° C., and 131 μl (1.02 mmol) ofbutyl chloroformate was added. After 30 min, the mixture free of theprecipitated triethylamine hydrochloride was pipetted in a solution ofO-phosphorylethanolamine (“Sigma-Aldrich”) (212 mg, 1,5 mmol) in 3 ml of1M Na₂CO₃ and 3 ml of H₂O. The mixture obtained was stirred for 1 h at20-25° C., acidified with 1M HCl to pH 2-3 and extracted withchloroform-methanol (2:1, v/v). The extract was washed withmethanol-water (10:9, v/v), concentrated under reduced pressure anddissolved in chloroform-methanol-NH₃ aq (13:5:1, v/v/v). The solutionobtained was evaporated under reduced pressure and dissolved inethanol-water (2:3, v/v, 15 ml). The emulsion obtained was washed withether (2×10 ml) and evaporated under reduced pressure gave the ammoniumsalt of N-acyl-O-phospho-2-aminoethanol.

Yields: 84% (5); 88% (9); 83% (13); 73% (17).

All the products obtained are identical to the compounds prepared by themethod using β-cyanoethyl phosphate for phosphorylation of the N-acylderivatives.

For ¹H-NMR-spectra ammonium salts were converted into acidified forms bymeans of washing of the chloroform-methanol (2:1, v/v) solutions with 1MHCl.

Example 18

Synthesis of The N-Retinoil-O-Phospho-L-Tyrosine

Were Retinoyl is:

all-trans-Retinoyl (4)

13-cis-Retinoyl (4a)

Retinoic acid (1 mmol) and triethylamine (142 μl, 1,02 mmol) weredissolved in 6 ml of tetrahydrofuran, the mixture chilled to −15° C.,and 131 μl (1.02 mmol) of butyl chloroformate was added. After 30 min,the mixture was added to the solution of

261 mg (1 mmol) of O-phospho-L-tyrosine (“Sigma-Aldrich”) in 3 ml of 1MNa₂CO₃ and 3 ml of H₂O Then 3 ml of EtOH was added. The mixture obtainedwas stirred for 4 h at 20-25° C., acidified with 1M HCl to pH 2-3 andextracted with chloroform-methanol (2:1, v/v). The extract was washedwith methanol-water (10:9, v/v), concentrated under reduced pressure anddissolved in chloroform-methanol-NH₃ aq (13:5:1, v/v/v). The solutionobtained was evaporated under reduced pressure and dissolved inethanol-water (2:3, v/v, 15 ml). The emulsion obtained was washed withether (5×10 ml) and evaporated under reduced pressure gave the ammoniumsalt of N-retinoyl-O-phospho-L-tyrosine.

Yields: 52% (4); 49% (4a).

All the products obtained are identical to the compounds prepared by themethod using β-cyanoethyl phosphate for phosphorylation of the N-acylderivatives.

For ¹H-NMR-spectra ammonium salts were converted into acidified forms bymeans of washing of the chloroform-methanol (2:1, v/v) solutions with 1MHCl.

The present invention further concerns therapeutically useful compoundsand the use of the same for the treatment of cancer. The invention alsorelates to the synthesis of amides of all-trans-retinoic acid and13-cis-retinoic acid with doxorubicin having the following structures:

1. N-(all-trans-retinoyl)-doxorubicin

2. N-(13-cis-retinoyl)doxorubicin

It is known that usefulness of natural retinoids for chemoprevention islimited by their toxicity. Synthetic retinamides that possesschemopreventive activity are less toxic then the natural retinoids(Shealy Y. F., et al., J. Med. Chem. V.31., P. 190-196, 1988). The titlecompounds could reveal not only the higher cytotoxicity to tumor cells,but a marked diminution in the toxicity of its preparations.

Our experimental results indicate that compound 1 reveals higherantitumor activity concerning Ehrlich ascites carcinoma (EAC), passed inC3H mice. Antitumor effect of compound 1 was dose-dependent. It isevidently, that compound 1 possess higher activity then that freedoxorubicin at equivalent dose. Injection of compound 1 at the dose 50mcg/mouse (2,5 mcg/kg) inhibited tumor growth by 57% while freedoxorubicin at the same dose inhibited EAC growth by 45%.

The higher cytotoxic effects the conjugates of polyunsaturated fattyacids with daunomicin to AFP-generating rat hepatoma cells could be dueto high affinity of AFP to arahidonic and docosahexaenoic acids. Thecomplex AFP with fatty acids, modified by daunomicin, was delivered toAFP-generating rat hepatoma cells, selectively.

Non AFP-producing EAC was used in our experiments. It is establishedthat reduce of antitumor activity of compound 1 is caused by thepresence of that protein (AFP) in the preparations, which were used forinjections to mice. The result of these experiments could be interpretedas a presence an equilibrium reversible complex formed between AFP andretinoic acid, modified by doxorubicin. Thus higher cytotoxic activityof compound 1, in comparison with doxorubicin, could be due to itsstructural peculiarities. The compound 1 include two antitumoragens—amide of retinoic acids and doxorubicin.

Example 19

Synthesis of N-(all-trans-retinoyl)-doxorubicin (compound 1)

all-trans-Retinoic acid (300 mg, 1 mmol) and triethylamine (104 mg, 1.02mmol) were dissolved in 1 ml of dry tetrahydrofluran, then dryacetonitrile (4 ml) was added, and the mixture chilled to −15° C. Then140 mg (1.02 mmol) of butyl chloroformate was added. After 30 min, themixture free of the precipitated triethylamine hydrochloride waspipetted in a stirred suspension of Doxolem preparation containing 400mg of doxorubicin hydrochloride and 2 g of lactose in a mixture of 1 mlof methanol and 0.1 ml triethylamine, stirring was continued for 15 minat −15° C., then the mixture obtained was allowed to warm to roomtemperature. After the mixture had stirred at room temperature underargon for 2 h, it was treated with 20 ml of benzene-ethanol (4:1). Thesuspension was centrifuged for 5 min at 3000 rpm, and the precipitatewas treated with 20 ml of benzene-ethanol (4:1) and harvested bycentrifigation (5 min, 3000 rpm). The two supernatants obtained werecombined and evaporated to dryness in vacuo. The residue was dissolvedin chloroform, and N-all-trans-retinoyl)-doxorubicin was purified byflash chromatography on silica gel (230-400 mesh). The column was elutedsuccessively with chloroform, acetone-chloroform (1:9, v/v),acetone-chloroform (1:4, v/v) and finally with benzene-ethanol (4:1).The pure product was eluted with benzene-ethanol (4:1) as a deep-redband. The solvent was evaporated in vacuo to give 445 mg (78%) ofN-(all-trans-retinoyl)-doxorubicin as a deep-red wax; TLC(benzene-dioxan-acetic acid 10:5:1) R_(f) 0.5 (for doxorubicine 0.08);UV (ethanol) A^(0.1%)(345 nm)=817; A^(0.1%) (497 nm)=196; ¹H-NMR (CDCl₃,200 MHz) δ 0.9-1.0 (t and s, 6H, 3 CH₂ ring in retinoic acid), 1.2-1.3(d, 3H, CH₃-5′), 1.4-2.4 (m, 19H, 5CH₃-RA, CH₂-2′ and CH₂-8), 2.9-3.3(q, 2H, CH₂-10), 3.7 (s, 1H, H-4′), 3.9-4.3 (m, 6H, OH-4′, OCH₃, H-3′,H-5′), 4.6 (s, 1H, COCH₂OH), 4.8 (s, 2H, COCH₂OH), 5.0-5.1 (d, 1H,OH-9), 5.3 (s, 1H, H-7), 5.5 (s, 1H, H-1′), 5.6-7.0 (m, 7H, 6HC═C-RA andNH), 7.3-7.4 (d, 1H, H-3), 7.7-7.8 (t, 1H, H-2), 8.0-8.1 (d, 1H, H-1),13.1 and 14.0 (two s, 2H, OH-6 and OH-11)

Example 20

The Antitumor Effect of N-(all-trans-retinoyl)-doxorubicin (compound 1)on EAC Cells

A study of the anti-tumour effect of compound 1 as compared withdoxorubicin was carried out in EAC, passed in C3H mice. 20-22 g males ofC3H mice were used. Control and experimental groups included 7 animals,which were inoculated 5×10⁶ EAC cells i.p. in volume 0.2 ml at day 0. Onsecond, fourth and sixth days following inoculation the doses of 20, 50and 100 μg/mouse of compound 1, equivalent ones of doxorubicin, in 200μl of normal mice serum (NMS) were injected i.v. Three groups of animalswere administrated water solutions of doxorubicin in doses_of 1, 2.5 and5 μg/kg of body weight (20, 50 and 100 μg/mouse by weight of 20 gaccordingly). One group of mice was untreated control. Results ofexperiment were accounted on ninth day. Mice were killed by cervicaldislocation. Ascitic fluids were removed, collected, their volumes weremeasured, and abdominal cavities were washed by saline solution 6-7times. Thus obtained fluids were pooled. The number of viable tumorcells was counted by hemocytometer, using trypan blue exclusion test.The mean and the standard mean error was calculated for each group ofmice. Comparison of tumor cell number in control and experimental groupswas carried out using Student t-test. Influence of tested preparation onEAC growth inhibition was evaluated by:${Inhibition},{\% = {\frac{{Control} - {Experiment}}{Control} \times 100}}$

The experimental results as shown in Table 1 indicate that compound 1displays high antitumor activity towards EAC. The anti-tumour effect ofcompound 1 is dose-dependent. Injection of compound 1 three timesrepeatedly on the second, fourth and sixth days following inoculation indose of 1 mg/kg (20 μg/mouse) gives an EAC growth inhibition of 47% ascompared with control untreated animals (p<0.01). It is evident, thatcompound 1 possess a higher activity than that of free doxorubicin in anequivalent dose. In case of compound 1 injection in dose of 2.5 mg/kg(50 μg/mouse) according to taken scheme the tumour growth inhibitionreached to 56% (p<0.01) while free doxorubicin at the same doseinhibited EAC growth of 45% (p<0.01). The smaller effect (EAC growthinhibition of 53%) was obtained in case of injection compound 1 at doseequivalent to 5 mg/kg (100 μg/mouse) of doxorubicin. It is seen, thatthe cytotoxic effect of free doxorubicin at this dose is the higher thanthat of compound 1.

Thus, the experiment shows that compound 1 at doses, corresponding to 1and 2.5 mg/kg of doxorubicin, exerts high anti-tumour effect towardsEAC, and further, that the cytotoxicity of compound 1 is the higher thanthat of free doxorubicin.

TABLE 1 Antitumor effect of compound 1 and doxorubicin at three timesrepeated i.v injections to mice with EAC (M ± m, n = 7) Dose, AscitesEAC growth Group of μg/mouse, volume, Tumor cell inhibition, % animalsPreparation i.v. ml quantity, × 10⁶ to control P Control Untreated —5.05 ± 0.85 1213.6 ± 137.96 — — Experiment 1 Compound 1 20 3.50 ± 1.30640.0 ± 78.04 47.3 <0.01 Experiment 2 Doxorubicin 20 2.85 ± 1.35  765.8± 127.52 36.9 <0.05 Experiment 3 Compound 1 50 2.10 ± 1.70  535.0 ±167.90 55.9 <0.01 Experiment 4 Doxorubicin 50 1.85 ± 1.10 662.1 ± 99.1745.4 <0.01 Experiment 5 Compound 1 100  3.30 ± 1.20 571.4 ± 66.96 52.9<0.002  Experiment 6 Doxorubicin 100  0.80 ± 0.60  350.0 ± 108.21 71.2<0.001 

Example 21

Effect of N-(all-trans-retinoyl)-doxorubicin (compound 1) with AFP onGrowth of EAC in Mice

The influence of compound lwith different amount of AFP on the growth ofEAC was studied. The experiments were carried out on inbred C3H micehaving initial weight of 20-23 g. The used tumor cell strain wassupported by i.p. passages to mice of the same line. At the beginning ofthe research (day 0) four groups, each of which contained 7 mice, wereformed. All of the animals were inoculated by tumour cells in salinesolution i.p. (5·10⁶ cells in 0,2 ml per a mouse).

The injections of different preparations of compound 1 and doxorubicinwith AFP were carried out on second, fourth and sixth days after i.p.implantation of EAC cells to mice. The first group of animals wasuntreated (negative control). The mice were injected by NMS. The mice ofsecond group were injected by compound 1 containing doxorubicin in thedose of 1 μg/kg of body weight (20 μg/mouse) i.v., NMS being used assolvent. The animals of third and fourth groups were injected the samedose of compound 1, the protein being added to NMS (2,5 and 5 μg/mouse,accordingly). On the ninth day after inoculation by EAC cells the micewere killed and ascitic fluids were collected and estimatedquantitatively. Tumor cells suspensions were prepared and aliquotes weremixed with equal volume of 0,1% trypan blue solution. The quantity oftumor cells were counted with hemocytometer.

Results of the experiments were evaluated by means of variationstatistics method using Student t-test. The values of inhibiting effectsof the preparations were represented as percentage in comparison withcontrol.

As seen from presented data of experiment (Table 2) compound 1 in doseof 1 mg/kg_(20 μg/mouse) inhibits the EAC growth of 45% (p<0,01). Thisis in concordance with data of previous experiment. In the same time theinfluence of compound 1 (20 μg/mouse), loaded by AFP (2,5 μg/mouse), islowered up to 33% (p<0,01) of tumor growth inhibition. Antitumor effectvalue of compound 1 with double content of AFP in injected solution(31%, p<0,02) is not changed essentially. It is seen that effect ofcompound 1 with AFP (2,5 and 5,0 μg/mouse) is near to one of freedoxorubicin.

TABLE 2 Effect of compound 1 in the dose of 1 mg/kg (20 μg/mouse) withAFP at three times repeated i.v. injections to mice C3H on growth of EAC(M ± m, n = 7) EAC Dose of growth AFP, Ascites Tumor cell inhibition,μg/ volume, quantity, × % to Treatment mouse ml 10⁶ control p 1. NMSNone 3.4 ± 1.01 2157.8 ± 207.6 — — 2. Com- None 2.0 ± 0.25 1185.7 ±266.2 45.0 <0.01 pound 1 3. Com- 2.5 3.0 ± 0.60 1442.9 ± 108.3 33.1<0.01 pound 1 + AFP 4. Com- 5.0 2.8 ± 0.65 1481.4 ± 171.3 31.3 <0.02pound 1 + AFP 5. Dox- None — —  36.9* <0.01 orubicin *see Table 1.

Example 22

The Anti-tumour Effect of N-(13-cis-retinoyl)-doxorubicin (compound 2)on Growth of EAC in Mice

Mice C3H of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 10 ml/kg of body weight or 200 μl/mouseby weight of 20 g) once every other day, three times (day 2, 4 and 6).Mice of control group received NMS. In two groups of doxorubicin(positive controls) mice received doxorubicin alone (in NMS) in doses of1 or 2.5 mg/kg of body weight (20 or 50 μg/mouse by weight of 20 g)accordingly. Mice of two test groups received the compound 2 in NMS,containing doxorubicin in doses of 1 or 2.5 mg/kg of body weight (20 or50 μg/mouse by weight of 20 g) accordingly. Three days later after thefinal treatment with test compound mice were killed by cervicaldislocation on day 9. Tumour cell number was counted and the extent ofinhibition of EAC growth in mice was evaluated.

Mice of control group had (975.4±81.37)×10⁶ EAC cells in abdominalcavity. In the group with doxorubicin alone in the dose of 1 mg/kg thenumber of tumour cells was (635.9±122.78)×10⁶ and EAC growth inhibitionof 34.8%, p<0.05. In mice of test group with compound 2, containingdoxorubicin in doses of 1 mg/kg, the number of tumour cells was(533.5±94.23)×10⁶ and EAC growth inhibition of 45.3%, p<0.01. In thegroup with doxorubicin alone in the dose of 2.5 mg/kg the number oftumour cells was (537.4±92.89)×10⁶ and EAC growth inhibition of 44.9%,p<0.01. In mice of test group with compound 2, containing doxorubicin indoses of 2.5 mg/kg, the number of tumour cells was (405.7±120.62)×10⁶and EAC growth inhibition of 58.4%, p<0.002.

Thus, the compound 2 in doses, corresponding to 1 and 2.5 mg/kg ofdoxorubicin, exerts high antitumor effect in respect to EAC, exceedingthe effect of doxorubicin alone.

Example 23

Synthesis of the N-(13-cis-Retinoyl)-Doxorubicin/Compound 2/

This compound was prepared as described above for compound 1, using 1mmol (300 mg) of 13-cis-Retinoic acid; yield 429 mg (75%).

TLC (benzene-dioxan-acetic acid 10:5:1) R_(f)0.5 (for doxorubicin 0.08);UV (ethanol) A^(0.1%) (347 nm)=817; A^(0.1%) (497 nm)=196; ¹H-NMR(CDCl₃, 200 MHz) δ 0.9-1.0 (t and s, 6H, 3 CH₂ ring in retinoic acid),1,2-2,4 (m, 22H, CH₃-5′, 5 CH₃-RA, CH₂-2′ and CH₂-8), 2.9-3.3 (q, 2H,CH₂-10), 3.7 (s, 1H, H-4′), 3.9-4.3 (m, 6H, OH-4′, OCH₃, H-3′, H-5′),4.6 (s, 1H, COCH₂OH), 4.8 (s, 2H, COCH₂OH), 5.0-5.1 (d, 1H, OH-9), 5.3(s, 1H, H-7), 5.5 (s, 1H, H-1′), 5.6-7.0 (m, 7H, 6HC═C-RA and NH),7.3-7.4 (d, 1H, H-3), 7.7-7.8 (t, 1H, H-2), 8.0-8.1 (d, 1H, H-1), 13.1and 14.0 (two s, 2H, OH-6 and OH-11).

Example 24

Anti-tumour Effect of DXR/(C4+C4a)+C5 Complexes

(C4+C4a):C5 molar ratios were equal to 1.2:1; 1.6:1; 1.9:1 and 2.5:1.C4:C4a molar ratio was equal to 3:2. DXR—3.5 mg/kg of body weight.C5—6.4 mg/kg of body weight.

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/(C4+C4a)+C5 complex.(C4+C4a):C5 molar ratio was equal to 1.2:1. Mice of second test groupreceived the DXR/(C4+C4a)+C5 complex. (C4+C4a):C5 molar ratio was equalto 1.6:1. Mice of third test group received the DXR/(C4+C4a)+C5 complex.(C4+C4a):C5 molar ratio was equal to 1.9:1. Mice of fourth test groupreceived the DXR/(C4+C4a)+C5 complex. (C4+C4a):C5 molar ratio was equalto 2.5:1. Two days later after the final treatment with the testcomplexes mice were killed by cervical dislocation on day 8. Tumour cellnumber was counted and the extent of inhibition of EAC growth in micewas evaluated.

Mice of control group had (768.7±102.8)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(465.1±62.8)×10⁶ and EAC growth inhibition of 39.5%, p<0.05. In mice offirst test group the number of tumour cells was (283.6±71.4)×10⁶ and EACgrowth inhibition of 63.1%, p<0.002. In mice of second test group thenumber of tumour cells was (218.3±65.3)×10⁶ and EAC growth inhibition of71.6%, p<0.001, and with reference to DXR group (positive control) EACgrowth inhibition of 53.1%, p<0.02. In mice of third test group thenumber of tumour cells was (299.7±73.6)×10⁶ and EAC growth inhibition of61.0%, p<0.002. In mice of fourth test group the number of tumour cellswas (405.1±81.5)×10⁶ and EAC growth inhibition of 47.3%, p<0.02.

Thus, antitumour activity of DXR/(C4+C4a)+C5 complexes at (C4+C4a):C5molar ratios 1.2:1; 1.6:1; 1.9:1 and 2.5:1 exceeds the effect of DXRalone.

Example 25

Anti-tumour Effect of DXR/(C4+C4a)+(C5+C9+C13) Complexes

(C4+C4a):(C5+C9+C13) molar ratios were equal to 1:1; 1:1.4; 1:1.8 and1:2.3. C4:C4a molar ratio was equal to 2:3. C5:C9:C13 molar ratios wereequal to 0.5:1:1. DXR—3.5 mg/kg of body weight. (C4+C4a)—8.15 mg/kg ofbody weight.

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received the DXR/(C4+C4a)+(C5+C9+C13)complex. (C4+C4a):(C5+C9+C13) molar ratio was equal to 1:1. Mice ofsecond test group received the DXR/(C4+C4a)+(C5+C9+C13) complex.(C4+C4a):(C5+C9+C13) molar ratio was equal to 1:1.4. Mice of third testgroup received the DXR/(C4+C4a)+(C5+C9+C13) complex.(C4+C4a):(C5+C9+C13) molar ratio was equal to 1:1.8. Mice of fourth testgroup received the DXR/(C4+C4a)+(C5+C9+C13) complex.(C4+C4a):(C5+C9+C13) molar ratio was equal to 1:2.3). Two days laterafter the final treatment with the test complexes mice were killed bycervical dislocation on day 8. Tumour cell number was counted and theextent of inhibition of EAC growth in mice was evaluated.

Mice of control group had (817.2±99.9)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(517.7±74.9)×10⁶ and EAC growth inhibition of 36.9%, p<0.05. In mice offirst test group the number of tumour cells was (326.1±92.8)×10⁶ and EACgrowth inhibition of 60.1%, p<0.01. In mice of second test group thenumber of tumour cells was (259.0±72.6)×10⁶ and EAC growth inhibition of68.3%, p<0.001, and with reference to DXR group (positive control) EACgrowth inhibition of 49.8%, p<0.05. In mice of third test group thenumber of tumour cells was (316.3±81.3)×10⁶ and EAC growth inhibition of61.3%, p<0.002. In mice of fourth test group the number of tumour cellswas (431.5±74.1)×10⁶ and EAC growth inhibition of 47.2%, p<0.01.

Thus, the anti-tumour activity of DXR/(C4+C4a)+(C5+C9+C13) complexes at(C4+C4a):(C5+C9+C13) molar ratios 1:1; 1:1.4; 1:1.8 and 1:2.3 exceedsthe effect of DXR alone.

Example 26

Antitumour Effect of DXR/(C4+C4a)+(C5+C9+C13+C17) Complexes

(C4+C4a):(C5+C9+C13+C17) molar ratios were equal to 1:1.3; 1:1.6; 1:2and 1:2.5. C4:C4a molar ratio was equal to 1:1. C5:C9:C13:C17 molarratios were equal to 1:1:1:1. DXR—3.5 mg/kg of body weight.(C4+C4a)—8.15 mg/kg of body weight.

Mice ICR of 20-22 g were inoculated intraperitoneally with 2×10⁶ viableEAC cells. Starting two days later (day 2) mice were injectedintravenously (in the volume of 2.5 ml/kg body weight) once every otherday, three times (day 2, 4 and 6). Mice of control group receivedvehicle. In the group of DXR mice received DXR alone in the dose of 3.5mg/kg. Mice of first test group received theDXR/(C4+C4a)+(C5+C9+C13+C17) complex (C4+C4a):(C5+C9+C13+C17) molarratio was equal to 1:1.3. Mice of second test group received theDXR/(C4+C4a)+(C5+C9+C13+C17) complex. (C4+C4a):(C5+C9+C13+C17) molarratio was equal to 1:1.6. Mice of third test group received theDXR/(C4+C4a)+(C5+C9+C13+C17) complex (C4+C4a):(C5+C9+C13+C17) molarratio was equal to 1:2. Mice of fourth test group received theDXR/(C4+C4a)+(C5+C9+C13+C17) complex (C4+C4a):(C5+C9+C13+C17) molarratio was equal to 1:2.5. Two days later after the final treatment withthe test complexes mice were killed by cervical dislocation on day 8.

Tumour cell number was counted and the extent of inhibition of EACgrowth in mice was evaluated.

Mice of control group had (738.1±73.9)×10⁶ EAC cells in abdominalcavity. In group with DXR alone the number of tumour cells was(431.1±57.4)×10⁶ and EAC growth inhibition of 41.6%, p<0.01. In mice offirst test group the number of tumour cells was (235.9±65.1)×10⁶ and EACgrowth inhibition of 68.0%, p<0.001, and with reference to DXR group(positive control) EAC growth inhibition of 45.3%, p<0.05. In mice ofsecond test group the number of tumour cells was (223.7±70.3)×10⁶ andEAC growth inhibition of 69.7%, p<0.001 and with reference to DXR group(positive control) EAC growth inhibition of 48.1%, p<0.05. In mice ofthird test group the number of tumour cells was (322.6±88.5)×10⁶ and EACgrowth inhibition of 56.3%, p<0.01. In mice of fourth test group thenumber of tumour cells was (417.2±69.7)×10⁶ and EAC growth inhibition of43.5%, p<0.01.

Thus, the anti-tumour activity of DXR/(C4+C4a)+(C5+C9+C13+C17) complexesat (C4+C4a):(C5+C9+C13+C17) molar ratios 1:1.3; 1:1.6; 1:2 and 1:2.5exceeds the effect of DXR alone.

Although the invention has been described with regard to its preferredembodiments, which constitute the best mode presently known to theinventors, it should be understood that various changes andmodifications as would be obvious to one having the ordinary skill inthis art may be made without departing from the scope of the inventionas set forth in the claims appended hereto.

What is claimed is:
 1. A compound having the following formula:


2. A method for the treatment of cancer, comprising administering N-(alltrans-retinol)-doxorubicin(I) to a mammal.
 3. A method for the treatmentof cancer, comprising administering N-(13-cis-retinoyl)-dororubicin(II)to a mammal.
 4. The method of claim 2 wherein N-(alltrans-retinoyl)-doxorubicin(I) is administered intravenously.
 5. Themethod of claim 3 wherein N-(13-cis-retinoyl)-doxorubicin(II) isadministered intravenously.